JSH-23 is an NF-κB inhibitor that inhibits NF-κB transcriptional activity (IC50=7.1 μM) but does not affect IκBα degradation. JSH-23 is an antioxidant with anti-inflammatory activity.

NF-κB:7.1 μM

METHODS: Bone marrow macrophage BMMs were treated with JSH-23 (0.78-200 µM) for 48-96 h. Cell viability was measured by CCK-8 assay.
RESULTS: JSH-23 had no detectable toxic effects at concentrations below 50 µM. [1]
METHODS: Macrophages RAW 264.7 harboring the reporter gene pNF-κB-SEAP-NPT were treated with LPS (1 µg/mL) and JSH-23 (1-30 µM) for 16 days, and SEAP expression was assayed to reflect NF-κB transcriptional activity.
RESULTS: JSH-23 inhibited LPS-induced SEAP expression in a dose-dependent manner by 23±3% at 3 µM, 68±3% at 10 µM, and 103±4% at 30 µM. JSH-23 inhibited NF-κB transcriptional activity. [2]

METHODS: To investigate the role in diabetic neuropathy, JSH-23 (1-3 mg/kg) was administered orally to streptozotocin-induced diabetic rats once daily for two weeks.
RESULTS: JSH-23 treatment significantly reversed nerve conduction and nerve blood flow deficits in diabetic animals. The treatment also partially corrected the reduced mechanical pain threshold.JSH-23 treatment inhibited nuclear translocation of the p65/p50 subunit in the sciatic nerve. [3]

Measurement of NF-κB transcriptional activity: Macrophages RAW 264.7 transfected stably with reporter plasmid of pNF-κB-SEAP-NPT are treated with 1 μg/ml LPS and/or sample for 16 hours. As the reporter, SEAP activity in the cell-free culture media is measured as followed. Single cell-derived stable transfectants are plated in 5 ml of T-25 flask, and the media is decanted 24 h later. At this time, cells are washed twice with phosphate-buffered saline, and incubations are initiated by addition of new media. Chemicals are added to the culture medium after 24 h of incubations. Aliquots (25 ml) of medium from a control or chemical-treated cultures are taken at 0, 3, 20, 24, 48, and 72 h, heated at 65°C for 5 min to eliminate the alkaline phosphatase activity, and used immediately or stored at -20°C. Mixtures consisting of dilution buffer (25 ml), assay buffer (97 ml), culture media (25 ml), and 4-methylumbelliferyl phosphate (MUP, 1 mM, 3 ml) in each well of the 96-well plates are incubated for 60 min in the dark at room temperature. Fluorescence emits the product of the SEAP/MUP is measured at 449 nm using a 96-well plate fluorometer after excitation at 360 nm.

Macrophages RAW 264.7 are incubated with various concentrations of JSH-23 compound for 24 h. The cells are treated with WST-1 solution and absorbance is measured at 450 nm.(Only for Reference)